Project 1: Determine the molecular basis of cell-selective pyrvinium action.
To determine how pyrvinium inhibits AR activity, we must first identify its cellular target. We created a pyrvinium analog bearing chemical modifications that will allow for the cross-linking of drug to protein target and the purification of the cross-linked protein(s), which will be identified using mass spectrometry. Putative targets will be validated using in vitro and genetic approaches. The identity of the target will inform studies to define the mechanism of pyrvinium action, which must account for its cell-selective activity. We are currently optimizing the cross-linking and purification strategies using a SILAC-based approach that we hope to be able to use to find the target of not only pyrvinium, but other lead compounds. Those with experience in these techniques are highly encouraged to apply to the lab.
Project 2: Exploit cellular cross-talk regulation of AR activity to treat prostate cancer. Genetic and chemical screens of our AR conformation change assay led to the identification of several cellular pathways that potentially regulate AR conformation and subsequent activity via cross-talk. These pathways include signaling through the alpha1-adrenergic receptor, the vitamin K epoxide reductase (VKOR), mTOR, and several growth factor receptors. We will investigate the biological connection between each of these pathways and AR using molecular biology and biochemical approaches and we will look for an association of each pathway with prostate cancer grade or progression using the extensive prostate cancer tissue bank at the City of Hope. Should the pathway appear to be involved in AR-dependent PCa, we will test this hypothesis in animal models of prostate cancer, and finally, pursue clinical trials in targeted prostate cancer patient populations, using molecules that target the cross-talk pathways, many of which are approved by the FDA for other indications. There are positions available to take the lead on several of these pathways.
Project 3: Identify potential SARMs in vitro and develop an in vivo model to test their tissue-selectivity.
AR is expressed in many different human tissues, and as such, controls diverse physiological processes and is often involved in human disease. There is a significant clinical need for drugs to treat AR-related diseases, but it has proven difficult to treat disease in one tissue without causing problems in others. For instance, systemic inhibition of AR prevents the growth of prostate cancer, but it also results in bone and muscle loss. Conversely, androgen replacement therapy is indicated for a number of conditions, but this treatment may promote the growth of prostate cancer. We need to find a way to selectively control AR activity to safely treat these diseases; however, we are hampered by a lack of understanding of AR selectivity at the molecular level. We hope to use the tools we have developed to study AR activity to uncover the mechanisms by which AR is selectively controlled in specific tissues and to develop novel selective AR modulators (SARMs).
To date, there is no effective in vitro screening tool to identify potential SARMs, and there is not an effective animal model to assess the tissue-selectivity of SARMs. We have utilized the AR conformation change assay as an in vitro screening tool to identify potential SARMs. By expressing the AR conformation reporter in cell lines derived from prostate cancer, muscle, bone, kidney, liver, and brain cells (all important sites of AR action), we can screen for small molecules (or genes) that either inhibit hormone-induced AR conformation change or induce conformation change in the absence of hormone in a cell-type selective fashion. We have identified many compounds (and genes) with cell-type specific agonist or antagonist activity. We would like to identify several more compounds that fit specific clinical profiles and begin to understand how this cell-type selectivity is achieved. We would also like to test the tissue-selectivity of these potential SARMs in an animal model. To this end, we are developing a rodent model to assess AR activity in all important AR-expressing tissues. AR activity will be judged using the transcription of AR-regulated genes in tissues of interest, as opposed to physiological measurements in a few select tissues. It is hoped that this approach will yield lead compounds for several clinical conditions, including prostate-selective AR inhibitors for prostate cancer and ‘prostate-safe’ AR agonists for androgen replacement therapy. This is a large, multi-faceted project that has the support of an RA and would be great project for a motivated postdoc.
Project 4: Examine the relationship between systemic testosterone levels and prostate disease.
The prostate is superbly sensitive to aging – almost no disease exists in young men, but nearly every male between 50-80 will present with benign prostatic hyperplasia (BPH) or prostate cancer (PCa). The prostate is also superbly sensitive to androgens – if you reduce androgens, you inhibit both BPH and PCa. Paradoxically, systemic androgen production wanes with age, even as the risk of prostate disease rises. The answer to this riddle may lie in the fact that the levels of androgens in the prostate tissue itself remain high, despite declining systemic levels. In fact, even with systemic levels of testosterone (T) reduced to below 10% of normal with androgen deprivation therapy, the levels of T and DHT in prostate cancer samples are about the same as in the prostates of healthy, young men. In these PCa samples, the genomic regions which encode AR and/or steriodogenic enzymes which synthesize androgens (such as 5-alpha reductase) are almost always amplified. This suggests that the prostate is transforming in such a way to maintain a functional level of T and DHT. In this selection process, a proliferative phenotype may arise and lead to BPH and PCa. This has enormous clinical implications. It may be possible that, instead of increasing the risk of BPH and PCa, exogenously administered T, applied at the right time, may reduce the incidence of these diseases by providing a source of androgens for the prostate, thereby obviating the need to transform to produce its own.
We would like to explore this hypothesis in a rodent model of prostate proliferative disease. We will examine the correlation between decreasing systemic T levels and incidence of BPH in rats, and determine if interventions with exogenous T affect the incidence of BPH. We will examine the molecular basis for the selection of a proliferative phenotype in the setting of declining systemic T levels, looking for chromosomal and transcriptional changes in the different cells of the prostate. If animal studies bear fruit, we will pursue a clinical trial in humans that will be significantly powered to test whether androgen replacement therapy can reduce the incidence of BPH and PCa (or prolong their onset), as several smaller clinical trials have suggested. This project has not yet started, and we are looking for a talented and dedicated individual to take the lead.
